Cytochrome P450 monooxygenases (P450s) play an important role in the metabolism of a variety of hydrophobic compounds. They are involved in the synthesis of steroids, fatty acids, vitamins, and other biological processes like the detoxification of xenobiotics (Maurer et al, 2003; Urlacher et Girhard, 2012). P450s catalyze a wide variety of reactions including hydroxylations, N-oxidations, N-, O- and S-dealkylations, sulfoxidations, deaminations, desulfurations, dehalogenations, peroxidations, N-oxide reductions, rearrangement reactions, C—C and C—O phenol couplings, cleavage of C—C bonds and others (Bernhardt et al, 1996; Bernhardt et al, 2006;).
The ability of P450s to catalyze the regio-, chemo- and stereospecific oxidation of a vast number of substrates reflects their biological roles and makes them important candidates for biotechnological applications.
Particularly, steroid hormones are widely used as anti-inflammatory, contraceptive and antiproliferative drugs. In mammals, the synthesis of these steroids starts with the side-chain cleaving reaction of cholesterol to pregnenolone. Pregnenolone serves as a basis for the production of further steroid hormones such as hydrocortisone (Szczebara et al, 2003) and great interests are associated with its industrial large scale conversion from low-priced substrates such as cholesterol and its plant-derived analogs.
However, side-chain cleaving reaction of cholesterol to pregnenolone is a limiting step in steroids overall process. In mammals, it is catalyzed by a membrane-bound CYP11A1 enzyme using adrenodoxin and adrenodoxin reductase as electron carriers.
Great efforts have been spent in order to reconstitute a sterol side-chain cleaving enzyme system in recombinant microorganisms such as Escherichia coli (Sakamoto et al., EP2386634) but it remains difficult to obtain a satisfactory level of enzymatic activity mainly because of the fact that CYP11A1 of mammalian origin is an insoluble membrane-associated enzyme and that CYP11A1 does not seem to properly fold in prokaryotic hosts.
A plasmidless derivative of B. megaterium DSM319 has proven to be a valuable host for co-expressing the prokaryotic cytochrome P450 CYP106A2 from B. megaterium ATCC 13368 with bovine adrenodoxin reductase (AdR), and adrenodoxin (Adx); it has been applied for the whole-cell conversion of hydrophobic acids with terpene structure such as the antiinflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA). In this work, the recombinant B. megaterium system was investigated in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368 and an E. coli whole-cell system. The prokaryotic cytochrome P450 CYP106A2 from B. megaterium ATCC 13368 is one of only a few cloned bacterial steroid hydroxylases. It was recently identified as the first reported bacterial P450 diterpene hydroxylase, which is able to carry out a one-step regioselective allylitic hydroxylation of abietic acid (Bleif et al 2012).
However, a microorganism which allows, as a whole-cell catalyst, to express and catalyze at high rates the bioconversion of substrates from cytochrome P450 monooxygenases of eukaryotic origin, such as the side-chain cleaving reaction of cholesterol to pregnenolone by insoluble CYP11A1, is still needed.